International Journal of Clinical Medicine, 2012, 3, 344-351
http://dx.doi.org/10.4236/ijcm.2012.35067 Published Online September 2012 (http://www.SciRP.org/journal/ijcm)
1
Evaluation of the Anti-Trypanosoma cruzi Effects of the
Antipsychotic Drug Levomepromazine
Inga Eva Stik Lange, Elisabeth Mieko Furusho Pral, Anahí Magdaleno, Ariel Mariano Silber*
Departamento de Parasitologia, Instituto de Ciências, Universidade de São Paulo, São Paulo, Brazil.
Received May 22nd, 2012; revised June 24th, 2012; accepted July 15th, 2012
ABSTRACT
Chagas disease, caused by the protozoan Trypanosoma cruzi, is a relevant parasitic disease in the Americas. Current
chemotherapy relies on Nifurtimox and Benznidazole, which present serious drawbacks, including high toxicity, low
efficiency and the emergence of resistant strains. In the present work, the perspectives of levomepromazine, a tricyclic
compound belonging to the family of phenotiazines with well-known properties as antipsychotics were evaluated as a
potential anti-T. cruzi drug. We show that this drug is able to inhibit the proliferation of epimastigotes (IC50 = 0.41 ±
0.01 mM) and to interfere with the infection of the host cells (IC50 = 0.34 ± 0.01 mM). Interestingly, the treatment with
levomepromazine affected the ability of metabolites such as glucose, proline and glutamate to fuel the recovery of epi-
mastigotes after being submitted to metabolic stress. These findings prompt levomepromazine as a promising leader
drug to obtain new trypanocidal activities.
Keywords: Chagas Disease; Phenothiazines; Trypanothione Reductase; Amino Acid Metabolism; Chemotherapy
1. Introduction
Trypanosoma cruzi, the causative agent for Chagas dis-
ease, a relevant health problem in most American coun-
tries, as it is broadly distributed from Mexico to southern
Argentina and Chile, where it is endemic, with 10 - 12
million people infected and an estimated 25 million peo-
ple at risk of acquiring the disease. Two drugs made
available almost four decades ago for patients are still the
only ones currently in use to treat Chagas disease: Nifur-
timox (Nf) and Benznidazole (Bn). Both drugs are highly
efficient in the acute phase of the disease; however, their
efficiency to treat the chronic phase, when most patients
are diagnosed, remains controversial. In addition, it is
worth mentioning that other serious drawbacks have been
reported for both drugs: their high toxicity, which in
some cases forces the interruption of treatment, and evi-
dence of emerging resistant strains. Taken together, these
facts constitute major reasons to search for new thera-
peutic alternatives [1].
Phenothiazines are a family of drugs related to the thi-
azine class of heterocyclic compounds [2]. Phenothiazi-
nes, which consist of a sulphur-containing tricyclic or-
ganic compound with the formula S(C6H4)2NH, with side
chains bound to the middle ring, have been commonly
used psychotropic drugs since the 1950s. This family of
compounds became well known and has been prescribed
up to the present because of their antipsychotic effects
[3]. Their action as dopamine blockers at D2 receptors
and their neuroleptic activity has been well documented
in the literature [4-7]. More recently, a large variety of
structurally modified versions of these drugs has been
introduced, broadening the spectrum of their targets. In
this sense, papers published in the last twenty years
showed antitumoural, antimicrobial, antiparasitic and
anthelmintic activities for phenothiazine derivatives (re-
viewed in [2]). The biological activities identified for
members with different substitutions at different posi-
tions, mainly 2 and 10, were particularly interesting to us.
For example, it was shown that compounds having a
methyl-thio substituent at position 10 and a fluorine sub-
stituent at position 2 (triflupromazine) have interesting
antimicrobial properties [2]. Variants of these drugs, such
as triflupromazine and chlorpromazine, have been active
against pathogenic amoebas like Naegleria fowleri,
Acanthamoeba culbertsoni and A. polyphaga [8]. Despite
unknown mechanisms of action, it was proposed that the
drugs have an antagonistic effect on the amoebal
calmodulins [9]. It was also proposed that chlorpromaz-
ine, a lipophilic variant, could act by modifying the
plasma membrane in some way, thereby diminishing the
viability of these organisms. Chlorpromazine also showed
inhibitory activity on the growth of Leishmania donovani
promastigotes [10], the fungus Candida albicans [11],
*Corresponding author.

Evaluation of the Anti-Trypanosoma cruzi Effects of the Antipsychotic Drug Levomepromazine 345
the bacteria Staphylococcus aureus [12], Mycobacterium
avium [13] and M. tuberculosis [14]. Shigella spp., Vi-
brio cholera and V. parahaemolyticus are among the
microorganisms reported to be sensitive to trifluop-
erazine. In the present work, the possible trypanocidal or
trypanostatic effect of Levomepromazine (LM), an anti-
psychotic drug belonging to the family of phenothiazines,
was investigated. LM was able to inhibit the growth of
epimastigotes in a dose-dependent way, (IC50 = 0.41 mM)
in the range of the doses reported in the literature for
most of the tests with these drugs (which approximately
range between 0.05 and 0.50 mM). Finally, it is worth
mentioning that since the first half of past century, phe-
nothiazines have been used safely in the treatment of
human infection by the helminth Enterobius vermicularis
[15].
In the present work, we report the anti-T. cruzi activity
of levomepromazine. We also show its ability to syner-
gise the deleterious effects of nutritional stress, meta-
cyclogenesis and host-cell infection.
2. Materials and Methods
2.1. Reagents
D-Glucose, L-proline, L-glutamate, rotenone, antimycin
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) were obtained from Sigma (St. Louis,
MO, USA). RPMI culture medium and foetal calf serum
were obtained from Cultilab (Campinas, SP, Brazil).
Levomepromazine (LM) (for chemical structure see Fig-
ure 1) (Togrel®) was purchased from Ivvax (Argentina).
All other reagents were from Amresco (Solon, OH,
USA).
2.2. Cells and Parasites
The Chinese Hamster Ovary cell line, CHO-K1, was rou-
tinely cultivated in RPMI medium (Gibco BRL) supple-
mented with 10% heat-inactivated foetal calf serum
(FCS), 0.15% (w/v) NaHCO3, 100 units/mL penicillin
and 100 μg/mL streptomycin at 37˚C in a humid atmos-
phere containing 5% CO2. Epimastigotes of T. cruzi, CL
strain, clone 14 [16], were maintained in the exponential
Figure 1. Chemical structure of levomepromazine (LM), a
phenothiazine derived compound.
growth phase by subculturing every 48 h in liver infu-
sion-tryptose (LIT) medium supplemented with 10%
FCS at 28˚C [17]. Metacyclic trypomastigotes were ob-
tained as previously described [18]. Briefly, 20 × 106
epimastigotes in the exponential growth phase were har-
vested by centrifugation (5000 × g for 10 min), washed
with PBS, and resuspended in TAU medium (190 mM
NaCl, 17 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 8 mM
phosphate buffer pH 6.0). After 1-h incu bation at 28˚C,
cultures were centrifuged (5000 × g for 10 min), resus-
pended in TAU-3AG (TAU supplemented with 10 mM
proline, 50 mM L-sodium glutamate, 2 mM L-sodium
aspartate and 10 mM D-glucose) and incubated for 96 h
at 28˚C. Culture-derived trypomastigotes were obtained
by infection of CHO-K1 cells with trypomastigotes, as
described previously [19]. Trypomastigotes were col-
lected in the extracellular medium from five days post-
infection. In all cases, parasites were counted in a
Neubauer chamber.
2.3. Growth Inhibition Assays
Epimastigote growth-inhibition assays were developed as
previously described [20]. Briefly, epimastigotes in the
exponential growth phase (approximately 5 × 107
cells/mL) were washed three times by centrifugation and
resuspended in PBS and then cultured in fresh LIT me-
dium supplemented with or without (controls) different
concentrations of LM ranging from 0.2 to 1.1 mM at
28˚C. The assays were carried out in 96-well plates by
inoculating epimastigotes in 200 μl of medium (2.5 × 106
cells/mL). Cell growth was estimated by absorbance
readings at 620 nm every day for ten days. The absorb-
ance was transformed into a cell density value (cells/mL)
using a linear calibration equation previously obtained
under the same conditions (R2 = 0.99551, p < 0.05). The
concentration of LM that inhibited 50% of parasite
growth (IC50) was determined in the exponential growth
phase (fifth day) by adjusting the effect (growth inhibit-
tion values) as a function of the drug concentration to a
classical sigmoidal equation. As a cell growth inhibition
control, growth curves in which 60 µM rotenone and 0.5
µM antimycin were added to the culture medium were
run in parallel for all experiments.
2.4. The Effect of LM Growth Inhibition under
Stress Conditions
To analyse the combined effect of LM at different pH
values and at different temperatures, the growth curves
were generated as described above, by adjusting the LIT
pH to the desired values or incubating the cultures at
28˚C (no stress), 33˚C or 37˚C. To evaluate the effect of
LM combined with nutritional stress conditions, 2 × 107
parasites/mL were washed and resuspended in 0.2 mM

Evaluation of the Anti-Trypanosoma cruzi Effects of the Antipsychotic Drug Levomepromazine
346
LM, in PBS (as a control) or PBS supplemented with 3
mM glucose, 3 mM proline, or 3 mM glutamate for 48 h
in Eppendorf tubes. Subsequently, cell viability was es-
timated using the MTT assay. To evaluate the combined
effect of LM and oxidative stress, 2.5 × 106 parasites/mL
in the exponential phase were maintained for 90 min at
28˚C in PBS or with 100 µM H2O2 in the presence or
absence of 0.2 mM LM. The cells were then collected by
centrifugation and resuspended in LIT medium, and after
5 days, the number of cells/mL was determined as de-
scribed previously [21,22].
extracellular medium on the fifth day and counted in a
hemocytometer [21].
2.7. Statistical Analysis
All experiments were made at least in triplicates. A
one-way ANOVA followed by Dunnet’s test was used
for statistical analysis. To analyse synergism between
two independent treatments, a two-way ANOVA was
performed as described previously [23]. A p value less
than 0.05 was considered statistically significant.
3. Results
2.5. Effect of LM on Mammalian Cell Viability
3.1. Evaluation of the Trypanocidal Effect of LM
CHO-K1 cells (5.0 × 105 cells/mL) were inoculated in
24-well plates in FCS-supplemented RPMI medium, as
previously described, in either the absence (control) or
presence of increasing concentrations of LM. The cell
viability was determined using the MTT assay as previ-
ously described, and the IC50 was obtained by fitting the
data to a typical dose-response sigmoidal curve [21].
To investigate the effects of LM on T. cruzi growth, 2.5
× 106 epimastigotes/mL were cultured in LIT medium
supplemented with different concentrations of the drug,
ranging from 0.2 to 1.1 mM. The growth of epimas-
tigotes in LIT supplemented with 6 μM rotenone and 0.5
μM antimycin as well as non-supplemented LIT were
used as positive and negative controls for growth inhibi-
tion, respectively. The cultures corresponding to non-
supplemented LIT showed typical growth curves reach-
ing the maximum values of cell density (86.0 × 106
cells/mL at day 9) when compared to the treated cultures
(which ranged between 69.0 and 2.2 × 106 cells/mL at
day 9) (Figure 2). As expected, the rotenone/antimycin-
treated cultures did not grow. A dose-response effect was
observed for the treated parasites, in which the effect of
the drug was significant for all evaluated concentrations
of LM on day 5 after treatment, when compared with LIT
2.6. Effect of LM on Trypomastigote Bursting
CHO-K1 cells were grown on cover slips (approximately
105 cells) and infected with 0.5 × 104 trypomastigotes in
RPMI medium supplemented with 10% FCS. After 3 h at
37˚C, free trypomastigotes in the medium were removed
by washing with PBS, and the infected cells were main-
tained at 33˚C in RPMI medium supplemented with 2%
FCS, with or without different concentrations of LM.
These concentrations of LM were not toxic to the mam-
malian cells. The trypomastigotes were collected in the
Figure 2. Growth curve of epimastigotes of Trypanosoma cruzi treated or not with LM at 28˚C and pH 7.5: : 0 mM (nega-
tive control), 1.1 mM (﬩), 700 μM (►), 500 μM (*), 400 μM (●), and 200 μM (▲). Positive control for growth inhibition (○)
was performed using 0.5 μM antimycin and 6 μM rotenone. Inset: dose response curves of epimastigote densities at different
LM concentrations on day 4 post treatment.

Evaluation of the Anti-Trypanosoma cruzi Effects of the Antipsychotic Drug Levomepromazine 347
controls. The IC50 value was determined to be 0.41 ±
0.01 mM (Figure 2, inset).
3.2. Interaction of LM with Stress Conditions
As mentioned above, T. cruzi circulates between two
different types of hosts, vertebrates and invertebrates.
Within each host type, this parasite goes through differ-
ent territories, meaning that these organisms are exposed
to different physical, physicochemical and metabolic
environments throughout their life cycle. Thus, they are
always exposed to a variety of natural stress conditions,
including nutritional, oxidative, pH and thermal stresses.
As a result, we were interested in evaluating the interact-
tion between the treatment of T. cruzi epimastigotes and
these stress conditions.
3.2.1. Oxidative Stress
To investigate the effect of LM on parasites under oxida-
tive stress, cultures were initially exposed to different
concentrations of H2O2 as a challenging agent.
Dose-response curves were obtained from these treat-
ments, establishing the IC50 as 0.10 mM H2O2. To evalu-
ate the possible interaction between both treatments, the
parasites were incubated for 90 min with the IC50 con-
centration of H2O2 (0.10 mM) in PBS and further incu-
bated in LIT medium supplemented or not with the IC25
concentration of LM (0.25 mM). The combined treat-
ment of LM and H2O2 showed a statistically significant
effect (p < 0.05), measured as a reduction in the capabil-
ity of parasite to growth at day 5 post-treatment. The
percent recovery after treatment for LM-treated cells was
45.00%; for H2O2-treated cells, it was 83.80%; and for
the combination of both treatments, it was 95.60% (p <
0.05) (Figure 3(a)).
3.2.2. Nutritional Stress
To analyse the effect of LM on parasites under nutria-
tional stress, cultures containing 20 × 106 cells/mL were
starved by 48 h incubation in PBS or in PBS supple-
mented with 3 mM proline, glucose or glutamate (PRO,
GLC and GLU, respectively). Samples of each culture
were concomitantly exposed or not (control) to a treat-
ment of 0.25 mM LM. As previously observed [21,22],
the presence of any of the three metabolites contributed
to maintain the viability of the cells, compared to those
starved in just PBS (Figure 3(b)). Each culture treated
with LM showed a significant diminution of viability
with respect to its corresponding non-LM-treated pair,
showing that LM interferes with the ability of proline,
glutamate and glucose to extend the survival of parasites
under nutritional stress (p < 0.05 for all paired compare-
sons). The combination of nutrients starvation and LM
treatment resulted in a synergistic diminution of the vi-
ability relative to the results observed in the presence of
any tested metabolite.
3.2.3. Thermal Stress
The viability of epimastigotes exposed to the simultane-
ous treatment of LM and thermal stress was also evalu-
ated. For thermal stress, three temperatures were chosen:
28˚C, the optimal temperature of growth for the insect
vector stage; 37˚C, the temperature of the mammalian
host; and 33˚C, the optimal temperature for the progres-
sion of in vitro infection of mammalian cells [19]. The
non-treated cultures behaved as previously observed,
with the maximum growth rate and maximum station-
ary-phase cell densities achieved at 33˚C rather than
28˚C (Figure 3(c)). As previously observed, both the
growth rate and stationary-phase cell density were the
lowest at 37˚C; however, these values were in the range
of the control (28˚C). If LM interacts with mechanisms
related to heat-shock resistance, then a significant change
in the IC50 values at different temperatures would be ex-
pected. However, the obtained results did not show sig-
nificant interactions between LM and thermal stress.
3.2.4. pH Stress
To evaluate the possible interaction of LM treatment
with different pH conditions, the parasites were initially
incubated in LIT with the pH adjusted to different values,
and the cell growth at day 5 was determined. Parasites
maintained at pH = 7.5 grew at rates comparable to those
of the controls (control curve Figure 1), while the most
extreme acidic pH evaluated significantly decreased the
cell density achieved in these conditions. Surprisingly,
parasites grown in slightly acidic conditions (pH = 6.5)
showed the maximum growth inhibition (Figure 3(d)).
Interestingly, when cultures grown in these conditions
were subjected to the IC25 of LM (0.25 mM), no signifi-
cant interaction was found between LM and acidic pH. In
fact, an additive effect is clearly observed for each condi-
tion, showing that this compound does not interfere with
the resistance to this variable.
3.3. The Effect of LM on Differentiation to
Infective Metacyclic Trypomastigotes
One relevant process involving environmental sensing in
terms of nutrient availability is the differentiation from
the non-infective epimastigote to the infective trypomas-
tigotes (a process called metacyclogenesis). Metacyc-
logenesis begins after the cells are exposed to a nutria-
tional stress, which naturally occurs in the terminal por-
tion of the digestive tube of the invertebrate host. Be-
cause LM treatment interacts synergistically with the
effect of nutritional stress, we concluded that LM could
also be interfering with metacyclogenesis. Epimastigotes
were incubated in TAU-3AG differentiation medium

Evaluation of the Anti-Trypanosoma cruzi Effects of the Antipsychotic Drug Levomepromazine
348
(a) (b)
(c) (d)
Figure 3. Response of epimastigotes at different stress conditions and LM treatment. (a) The parasites were submitted to
oxidative stress by incubation in the presence of 0.10 mM H2O2 and the addition or not of drug (0.25 mM LM). The effect of
treatments and controls were measured by growing the parasites in LIT for five days and quantifying the cell density; (b) The
parasite viability was measured using the MTT assay after nutritional stresses performed by 72 h incubation in PBS, PRO,
GLU or GLC, combined or not with the drug treatment (0.25 mM LM); (c) The parasite density was measured at the 4th day
of growth, submitted or not to thermal stress (33˚C and 37˚C) and drug treatment (0.25 mM LM); (d) The parasite density
was measured at the 4th day of growth, submitted or not to pH stress (pH 5.5 or 6.5) and drug treatment (0.25 mM LM).
supplemented with or without (control) 0.41 mM
LM.The number of metacyclic forms was counted at 48,
72 and 96 h post-treatment. Because the treatment was
performed with the IC50, in the absence of any type of
interference with this differentiation process, a 50% de-
crease in the quantity of metacyclic forms for the treated
cells with respect to controls would be expected due to
50% death. Interestingly, metacyclics were inhibited by
71.47%, 80.00% and 88.79% at 48, 72 and 96 h post-
treatment, respectively (Figure 4), clearly showing that
LM diminishes the ability of epimastigotes to differenti-
ate into metacyclic trypomastigotes.
3.4. Cytotoxicity and Effect of LM on
Trypomastigotes Bursting from Infected
Host Cells
To evaluate the effects of LM on the treatment of in-
fected cells, the toxicity of LM on CHO-K1 cells was
Figure 4. The effect of LM on parasites differentiation.
Epimastigotes were incubated for different times (48, 72 or
96 h) in TAU-3AG differentiation medium for metacy-
clogenesis in the presence of 0 (Control) mM of LM (white
bars), 0.3 mM of LM (grey bars), or 0.4 mM of LM (black
bars). Metacyclic forms were counted by microscopical
observation in a Neubauer chamber.

Evaluation of the Anti-Trypanosoma cruzi Effects of the Antipsychotic Drug Levomepromazine 349
initially evaluated. The cells were incubated for 48 h in
culture medium supplemented with or without (control)
LM at concentrations ranging between 0.2 and 1.2 mM.
The IC50 was calculated to be 0.86 mM (Figure 5). De-
spite this value being close to the IC50 obtained for T.
cruzi epimastigotes, the effect of LM on the trypomas-
tigote bursting from the infected cells was evaluated.
Cells (1 × 106/well) were infected with 0.5 × 106 cul-
ture-derived trypomastigotes and incubated with medium
supplemented with or without (control) LM in a range of
concentrations between 0.1 and 0.6 mM. Interestingly,
the treatment with 0.5 mM LM reduced the trypomas
tigote bursting by more than 96%, resulting in an IC50 of
0.34 mM (Figure 6).
Figure 5. Evaluation of the cytotoxic effect of LM on
CHO-K1 cells. CHO-K1 cells were challenged with different
concentrations of LM, and the cell viability was measured
using the MTT assay.
Figure 6. The effect on the trypomastigotes bursting of the
treatment with LM of infected cells. The trypomastigotes
that bursted on the 7th day post-infection in cell cultures
treated or not with different concentrations of LM were
counted.
4. Discussion
As mentioned, phenotiazines are compounds that belong
to a large family of molecules with increasing prospects
for neuropathology therapy, cancer, immunomodulation
and infections [2,3]. As T. cruzi circulates throughout
different environments (different regions of the digestive
tube in the insect vector, or intra- and extracellular me-
dium in the mammalian host), it is naturally exposed to
several stresses in its life cycle [24]. Among these natural
stresses, oxidative, nutritional, thermal and pH stress are
relevant. Several vital mechanisms related to the ability
of T. cruzi to cope with these conditions have been de-
scribed [25]. The interference of LM with such mecha-
nisms could be relevant to prospect an in vivo study of
LM by itself or in combination with other drugs. No in-
terference of LM with oxidative, thermal or pH stress
was found in the present study. However, LM interfered
with the ability of the parasites to recover from nutria-
tional stress. The ability of proline, glutamate, aspartate
and glucose to delay the effect of parasite starvation was
previously shown [22]. Interestingly, LM interfered with
this ability when each of the aforementioned metabolites
was evaluated. This set of results strongly suggests that
LM is interfering with T. cruzi metabolism in a way that
affects its ability to use the tested carbon sources.
When the parasites were simultaneously submitted to
LM and oxidative, pH and thermal stress, no interactions
were observed. It is interesting the fact that, in spite of
being considered the optimal temperature as being 28˚C,
our experiments show that in our system, the optimal
temperature was 33˚C. This fact was previously observed
by us and described as being probably strain dependant
[21,22]. Starvation leading to nutritional stress occurs
when the parasite changes its environment throughout its
life cycle [26]. Particularly, it is well known that nutrient
starvation occurs in the transit from the medium to the
terminal portion of the insect gut [18]. In the rectum,
some nutrients are available due to their back-flow to the
digestive tube from the hemolymph through the Mal-
pighian tubules to the rectum. The most relevant nutri-
ents are precisely proline, glutamate and aspartate [18],
which are able to recover the intracellular ATP levels
that fuel metacyclogenesis [27]. Our results showed that
the ability of these nutrients to keep the epimastigotes
viable was abolished by treatment with LM. This finding
supported the hypothesis that LM could also interfere
with metacyclogenesis when induced by these metabolic
substrates. This rationale led us to evaluate the meta-
cyclogenesis gated by TAU-3AG in the presence or ab-
sence of LM. The results obtained confirmed that meta-
cyclogenesis was diminished by half in these conditions;
thereby supporting the idea that LM interferes with the
metabolic pathways that energetically support the intense
transformation experienced by these cells through meta-

Evaluation of the Anti-Trypanosoma cruzi Effects of the Antipsychotic Drug Levomepromazine
350
cyclogenesis.
Finally, the effect of LM on trypomastigote burst was
also evaluated. Our results showed that the treatment of
infected cells with 0.5 mM LM resulted in more than
90% inhibition of trypomastigote bursting. The treatment
of T. cruzi with another phenothiazine derived drug, thi-
oridazine (usually prescribed as a neuroleptic drug), at
low doses seemed to be promising for epimastigotes
(IC50 = 5 μM). However, trypomastigotes were less sen-
sitive to the drug, showing an IC50 of 0.5 mM [28]. In
addition, the treatment of mice with thioridazine partially
prevented the development of parasitemia; however,
some cardiac damage was not prevented, even in the ab-
sence of the evidence of amastigote nests [29].
Collectively, our data support the idea that the meta-
bolic steps involved in the oxidation of glucose, proline,
aspartate and glutamate are involved in the antiparasitic
activity of LM. Further experiments will be conducted to
define the molecular targets involved in this antiparasitic
activity, which will, in turn, lead to the optimisation of
more efficient LM-derived drugs.
5. Acknowledgements
The authors are deeply acknowledged to Dr. Laura Tanga
for helpful discussions and interesting suggestions. Fi-
nancial Support: this work was supported by grants from
the Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP grant #11/50631-1 to AMS), Instituto
Nacional de Biologia Estrutural e Química Medicinal em
Doenças Infecciosas (INBEQMeDI), and Conselho Na-
cional de Desenvolvimento Científico e Tecnológico
(CNPq grant # 470272/2011-2 to AMS).
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